Therapeutic treatments of human cancer using immune intervention are assuming a role of increasing importance Fahey et al., Ann. Intern. Med. (1987)) 106:257-74. These therapies include the use of lymphokines and lymphokineactivated effector cells, monoclonal antibodies and interferons. Additionally, certain types of immune-effector cells exhibit spontaneous cytotoxicity in the absence of specific lymphokine stimulation. (Rabinowich et al., Cancer Research 47:173-177 (1987)).
Immune intervention, at present, elicits a positive response in only about 1 out of 3 subjects even in cancers which are most successfully treated using this approach. It is evident that a tool for predicting the success or failure of a proposed biological response modification protocol would be of value in preventing unneeded suffering in individuals from ineffective treatment and in selecting from among various alternative protocols that which has the highest likelihood of success.
One approach to an in vitro method for prediction of cytotoxicity is based on the observation that immune-mediated cell damage includes disruption of membrane integrity which leads to cell lysis (Kimber and Moore, Exp. Cell Biol. 53:69-84 (1985)). Therefore it would appear that immune-mediated cell damage caused by immune-effector treatment could be based upon a determination of membrane integrity. The two most widely used assays having indications of membrane damage as endpoints are various dye exclusion and chromium 51 release assays (Endo et al., Cancer Research 47:1076-80 (1987); Gambacorti-Passerini et al., Cancer Research 47:2547-2552 (1987)). Dye exclusion assays are based on the ability of viable cells in the presence of a physiological salt solution, to exclude a dye which is taken up by cells killed through membrane lysis, so that cell death may be quantified in vitro. (See, e.g. Durkin et al., Cancer Res. 39:402-407 (1978)). Dye exclusion assays are typically performed by counting preparations of cells in the presence of a dye such as trypan blue, eosin, erythrocin-B, Fast-Green or nigrosin. Living cells, but not dead cells, exclude the dye. The chromium 51 release assay uses the radioactive substance chromium 51 to label cells, for example, tumor cells; Cr.sup.51 release indicates damage to the cell membrane.
Both of the foregoing assays yield reasonable results when the cell populations to which they are applied are homogeneous. However, non-tumor cells (macrophages, lymphocytes, mesothelial cells and other normal elements) frequently outnumber the tumor cells which are present in human neoplasms. In diseases such as multiple myeloma and acute leukemia the neoplastic cells may only represent 10% of the total cell population present. Standard dye exclusion assays, whether based on trypan blue and light microscopy or propidium iodide and flow cytometry suffer from several disadvantages, including poor ability to discriminate between effects on tumor and non-tumor cells. Additional difficulties with standard dye exclusion assays have been described ((Weisenthal et al., Cancer Res. 43:258-264 (1983)). The Cr.sup.51 assay also fails to distinguish between normal and tumor cells.
In addition, there is a great deal of spontaneous chromium 51 release by cells, whether alive or dead, occasionally exceeding 50% over a three hour period. Thus, it is very difficult to determine with precision how much chromium release is due to the drug and how much is caused by damage to tumor cells as opposed to non-tumor cells. Finally, because of the rapid spontaneous release, the duration of the assay is constrained to a period of only several hours. This mandates the use of large concentrations of the immune effectors.
An in vitro assay (the differential staining cytotoxicity or "DiSC" assay) which permits prediction of the chemosensitivity of human tumors to various drugs, without requiring tumor growth in culture, has been reported (Weisenthal et al., Cancer 51:1490-1495 (1983); and Weisenthal et al. in Recent Results in Cancer Research, 94:161-173, Springer Verlag, Berlin-Heidelberg (1984)). The DiSC assay, further described below as readily adaptable to the method of the present invention, permits discrimination of cytotoxic effects between tumor and non-tumor cell populations co-existing in the same cell suspension. The co-existence may be advantageous, since it more realistically mimics the situation in vivo. This assay relies on the ability of living cells to exclude certain dyes such as Fast Green, Nigrosin or the combination of Fast Green and Nigrosin, while dead cells cannot. Living cells may be counterstained, for example with Wright Giemsa (hematologic neoplasms) or hematoxylin and eosin stain (solid tumors) to facilitate counting. The number of individual cells capable (or not) of dye uptake is ascertained by a technician counting cell populations using a microscope. Thus the assay provides a means for individually counting the number of tumor cells surviving in the presence or absence of a chemotherapeutic agent when the biopsied tumor cells are subjected to the assay. Methods for monitoring human patients for acquisition of resistance to chemotherapeutic drugs and for designing alternative programs of chemotherapy for tumors which become resistant to the drug using the DiSC assay, have been reported (Weisenthal et al., Cancer Treat Rep. 70:1283-1295 (1986); and Weisenthal et al., Cancer Treat Rep., In Press, Nov. 1987).
Immune therapies are very expensive, frequently toxic and only sporadically effective. It would thus be beneficial to provide a method for in vitro prediction of the probable clinical efficacy of immune effector substances and the ability to thereafter monitor patients for acquisition of resistance to such therapies, as well as to design modification of treatments to overcome such resistance. The method could be used to determine the most promising strategies of immune interventions in cancer and other disease states. Such a method is provided by the invention herein.